The goal of this project is to expand our understanding of how cells in the blood vessel wall transduce the physical force shear stress. Shear stress is the frictional force exerted on the vessel wall due to the flow of blood through the lumen, and is the primary stimulus that governs both acute changes in blood vessel diameter and chronic structural remodeling. These processes are critically important for ensuring appropriate blood flow to tissues, which is essential for human health. Remarkably, how the cells in the vessel wall transduce the shear stress into a dilation response or structural remodeling is not clearly understood. Therefore, the overall objective of this project is to uncover key molecular signaling events that control the vascular response to shear stress, with a focus on endothelial nitric oxide synthase (eNOS). eNOS is a key enzyme critical to blood vessel dilation and vascular remodeling. Uncovering its regulatory mechanisms during shear stress will be achieved through two specific aims. Aim 1 is to determine how eNOS is regulated acutely by shear stress in intact arteries, through examining key regulatory phosphorylation sites on eNOS (Tyr83, Ser116, Thr497, Ser617, Ser635, Tyr657, Ser1179), the role of upstream kinases, protein-protein interactions (caveolin-1, Hsp90), and concomitant diameter changes, using an isolated, cannulated, perfused mouse artery preparation and video microscopy. Arteries (mesenteric and carotid arteries) will be subjected to various magnitudes of shear stress (with or without specific kinase inhibitors), at sequential time-points, and analyzed for phosphorylation and protein-protein interaction using immunoblotting and immunoprecipitation. These experiments will illuminate how this enzyme is regulated during shear stress-induced dilation. Aim 2 is to determine how eNOS is regulated during chronically elevated shear stress in intact arteries, by examining eNOS phosphorylation and protein-protein interactions during the remodeling process. This will be measured: 1) in vitro using an innovative method in which isolated, cannulated mouse arteries are cultured for several days in a chronic perfusion system, and 2) in vivo using a surgical ligation model to chronically increase flow in the external carotid artery and the small mesenteric arteries of a mouse. These experiments will provide insight into when and how eNOS is activated during chronic shear stress-induced remodeling, which is currently unknown. PUBLIC HEALTH RELEVANCE: This project will examine, at the molecular level, how arteries respond to the physical force produced by blood flowing through the artery. This force influences both the structure and the health of the artery. Healthy arteries are critical to overall human health because when they are unhealthy, atherosclerosis and heart disease results.